Inhibition of activated cdc42-associated kinase 1

ABSTRACT

Compounds, compositions, and methods for specific inhibition of activated cdc42-associated kinase 1 (Ack1) are provided.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.13/429,755, which was filed on Mar. 26, 2012, and claims priority toU.S. Provisional Application No. 61/467,013 filed on Mar. 24, 2011, thecontents of each application are incorporated by reference herein, intheir entirety and for all purposes.

REFERENCE TO A SEQUENCE LISTING

This application includes a Sequence Listing submitted electronically asa text file named Ack1 Sequence Listing_ST25.txt, created on Mar. 26,2012, with a size of 1,000 bytes. The Sequence Listing is incorporatedby reference herein.

FIELD OF THE INVENTION

The invention relates generally to the field of formulation chemistry.More particularly, the invention relates to compounds, compositions, andmethods for specifically inhibiting activated cdc42-associated kinase 1(ACK1) and for treating ACK1-associated diseases including, for example,breast, ovarian, pancreatic, lung and prostate cancers and L-dopainduced dyskinesia.

BACKGROUND OF THE INVENTION

Various publications, including patents, published applications,technical articles and scholarly articles are cited throughout thespecification. Each of these cited publications is incorporated byreference herein, in its entirety and for all purposes.

Protein kinases are among the important classes of therapeutic targetsbecause of their central roles in cell signaling pathways. The presenceof a highly conserved ATP-binding site in kinases consists of a deephydrophobic pocket, adapted for small molecule binding, that can beexploited by agents. Due to the evolutionary conservation of this pocketbetween protein kinases, however, achieving highly selective kinaseinhibition by ATP-competitive inhibitors is a significant challenge. Indrugs, these off-target activities can produce dose-limiting toxicitiesthat limit therapeutic efficacy. In research compounds, off-targetactivities confound experimental interpretation. Thus, defining thespecificity of kinase inhibitors using broad panels of diverse proteinkinases is important.

Knowledge of target selectivity for kinase inhibitors is important forpredicting and interpreting the effects of inhibitors in both theresearch and clinical settings. Recent technological advances have ledto the development of several methods to profile kinase targetselectivity against significant fractions of the 518 human proteinkinases. These include kinase-inhibitor binding (or displacement)assays, cell-based profiling methods, and high-throughput enzymaticassays. Initial applications of these methods have revealed a strikingdegree of promiscuity of these compounds, even those thought highlyspecific. Off-target inhibition is frequently observed even of kinasesonly distantly related to the primary target. These findings haveemphasized the importance of comprehensive testing of kinase inhibitorspecificity.

Generally, kinase inhibitors have been identified in a target-centricmanner in which inhibitors are developed through an iterative processagainst a particular kinase of interest. The resulting compounds arethen tested for specificity against a panel of representative kinases.An alternative approach has been suggested in which large libraries ofcompounds are initially screened in parallel against comprehensivepanels of recombinant protein kinases. Compounds showing desiredselectivity patterns are then chemically optimized for the desiredtarget(s). The cost of implementing this strategy for very largecompound libraries, however, is prohibitive.

SUMMARY OF THE INVENTION

The invention features compounds, for example, a compound of Formula I:

or a pharmaceutically acceptable salt thereof,wherein each of R₁ and R₂ is selected from the group consisting of:R₄—O—, H, and

and

R₃ is

wherein R₄ is a C₁-C₆ alkyl or H;

-   -   R₅ is a C₃-C₈ cycloaklyl such as cyclopropyl, benzyl,

and

-   -   R₆ is F, Cl, or OMe; and    -   R₇ is a C₁-C₃ alkylene.

In preferred embodiments, R₁ is H₃CO—, R₂ is a morpholinopropoxy group,and R₃ is

where R₅ is a benzyl group or a substituted benzyl group. In someaspects, when R₁ is H₃CO— and R₂ is morpholino, R₃ is not cyclopropyl or

where R₇ is methyl, and when R₁ and R₂ are each hydrogen, R₃ is not

where R₅ is benzyl. The compound of Formula I may be comprised in acomposition with a carrier such as a pharmaceutically acceptablecarrier.

The invention also features methods for inhibiting the biologic activityof activated cdc42-associated kinase I (Ack1). In some aspects, themethods comprise contacting Acid with an effective amount of a compoundof Formula I, or a composition comprising a compound of Formula I and acarrier. Preferably, the compound is capable of inhibiting the biologicactivity of Acid at an IC₅₀ of about 5 nM or less.

In some aspects, the methods comprise contacting Acid with an effectiveamount of a compound of Formula II:

or a pharmaceutically acceptable salt thereof,wherein R₈ is selected from the group consisting of:

andR₉ is selected from the group consisting of:

wherein R₇ is a C₁-C₃ alkyl; and X is F, Cl, I, Br, or H. In someaspects, the methods comprise contacting Ack1 with an effective amountof a composition comprising a compound of Formula II and a carrier.Preferably, the compound is capable of inhibiting the biologic activityof Ack1 at an IC₅₀ of about 5 nM or less.

In some aspects, the methods comprise contacting Ack1 with an effectiveamount of a compound of Formula III:

or a pharmaceutically acceptable salt thereof,wherein R₁₀ is selected from the group consisting of:

and R₁₁ is selected from the group consisting of:

benzyl and propyl, wherein R₇ is a C₁-C₃ alkyl; and X is F, Cl, I, Br,or H. In some aspects, the methods comprise contacting Ack1 with aneffective amount of a composition comprising a compound of Formula IIIand a carrier. Preferably, the compound is capable of inhibiting thebiologic activity of Ack1 at an IC₅₀ of about 5 nM or less.

The invention also features methods for inhibiting the biologic activityof activated cdc42-associated kinase I (Ack1) in a cell. In someaspects, the methods comprise contacting a cell expressing Ack1 with aneffective amount of a compound of Formula I, or a composition comprisinga compound of Formula I and a carrier. In some aspects, the methodscomprise contacting a cell expressing Ack1 with an effective amount of acompound of Formula II, or a composition comprising a compound ofFormula II and a carrier. In some aspects, the methods comprisecontacting a cell expressing Ack1 with an effective amount of a compoundof Formula III, or a composition comprising a compound of Formula IIIand a carrier. Preferably, the compound is capable of inhibiting thebiologic activity of Ack1 at an IC₅₀ of about 5 nM or less. The cell maybe a cancer cell such as a prostate cancer cell, a breast cancer cell, alung cancer cell, a pancreatic cancer cell, an esophageal cancer cell,or a squamous cell carcinoma cancer cell, or may be a neuron.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows a pie chart showing the representation of different kinasefamilies for the intended targets for the kinase inhibitors in thescreening library. FIG. 1B shows the distribution of kinases tested inthe kinase panel. Dots representing individual protein kinases in thepanel are shown on a dendrogram representing the complete human kinome(based on amino acid sequence similarity between kinases within thecatalytic domain). FIG. 1C shows a scatter plot representing thereproducibility of the screening data. Each point represents onekinase-inhibitor pair plotted as the residual kinase activity in onereplicate versus activity in the second replicate, for allkinase-inhibitor pairs in which at least 20% inhibition was observed.

FIG. 2A shows the catalytic activity of 300 kinases in the presence of500 nM 1; average of duplicates are shown. FIG. 2B shows a dose-responseof Formula I compound against Ack1.

FIG. 3 shows the structure of compound 1 (Formula I) and sample analogs.Compound 1 has an IC₅₀ of 5 nM in vitro. Compound 1.1 has similarpotency and inhibits Ack1 in cells (FIG. 5).

FIG. 4 shows the synthesis of compounds 1 and 1.1 by an in house organicsynthesis facility.

FIGS. 5A and 5B show that the Formula I compound inhibits Ack1autophosphorylation in cells and has high target specificity. FIG. 5Ashows results of a Western blot for phospho-Ack1; HEK293 cellsexpressing Ack1 were treated for 15 min with Formula I, and cell lysatewas analyzed. FIG. 5B shows the kinase specificity of Formula I againstthe panel of 300 protein kinases as in FIG. 2A.

FIG. 6 shows the structure of compound 2 (Formula II) and analogs.

FIG. 7 shows the structure of compound 3 (Formula III) and analogs.

DETAILED DESCRIPTION OF THE INVENTION

Various terms relating to aspects of the invention are used throughoutthe specification and claims. Such terms are to be given their ordinarymeaning in the art, unless otherwise indicated. Other specificallydefined terms are to be construed in a manner consistent with thedefinition provided herein.

As used herein, the singular forms “a,” “an,” and “the” include pluralreferents unless expressly stated otherwise.

Subject and patient are used interchangeably. A subject may be anyanimal, including mammals such as companion animals, laboratory animals,and non-human primates. Human beings are preferred.

Inhibiting comprises reducing, decreasing, blocking, preventing,delaying, inactivating, desensitizing, stopping, and/or downregulatingthe biologic activity or expression of a molecule or pathway ofinterest.

It has been observed in accordance with the invention that certaincompounds can inhibit the biologic activity of activatedcdc42-associated kinase 1 (Ack1) without substantially inhibiting thebiologic activity of other kinases, or at least can inhibit the biologicactivity of Ack1 to a greater extent than other kinases, and with abouta 5 nM IC₅₀. Selective inhibition of Ack1 has implications for treatmentof certain types of cancers as well as L-dopa induced dyskinesia (LID)and other diseases, disorders, or conditions that are caused by,facilitated by, exacerbated by, or otherwise involve biochemicalpathways modulated or regulated by the biologic activity Ack1.Accordingly, the invention features compounds, compositions, and methodsfor inhibiting the biologic activity of Ack1.

The invention features methods for inhibiting the biologic activity ofactivated cdc42-associated kinase I (Ack1). The methods may be carriedout in vitro, in vivo, or in situ.

In some aspects, the methods comprise contacting Ack1 with an effectiveamount of a compound having Formula I:

or a pharmaceutically acceptable salt thereof,wherein each of R₁ and R₂ is selected from the group consisting of:R₄—O—, H, and

and

R₃ is

wherein R₄ is a C₁-C₆ alkyl or H;

-   -   R₅ is a C₃-C₈ cycloalkyl such as cyclopropyl, benzyl,

and

-   -   R₆ is F, Cl, or OMe; and    -   R₇ is a C₁-C₃ alkyl.

In preferred embodiments, R₁ is H₃CO—, R₂ is a morpholinopropoxy group,and R₃ is

where R₅ is a benzyl group or a substituted benzyl group.

In some aspects, the methods comprise contacting Ack1 with an effectiveamount of a compound having Formula II:

or a pharmaceutically acceptable salt thereof,wherein R₈ is selected from the group consisting of:

andR₉ is selected from the group consisting of:

wherein R₇ is a C₁-C₃ alkyl; and X is F, Cl, I, Br, or H.

In preferred embodiments, R₈ is a sulfur-containing group, R₉ is ahalogen-substituted benzyl group wherein the halogen is a fluoro group.

In other aspects, the methods comprise contacting Ack1 with an effectiveamount of a compound having Formula III:

or a pharmaceutically acceptable salt thereof,wherein R₁₀ is selected from the group consisting of:

and R₁₁ is selected from the group consisting of:

benzyl and propyl, wherein R₇ is a C₁-C₃ alkyl; and X is F, Cl, I, Br,or H.

Preferably, the biologic activity of Ack1 is inhibited by the compoundwith less than about a 100 nM IC₅₀, more preferably inhibited by thecompound (Formula I, H, and/or HI) with less than about a 50 nM IC₅₀,more preferably inhibited by the compound with less than about a 40 nMIC₅₀, more preferably inhibited by the compound with less than about a30 nM IC₅₀, more preferably inhibited by the compound with less thanabout a 20 nM IC₅₀, more preferably inhibited by the compound with lessthan about a 10 nM IC₅₀, more preferably inhibited by the compound withless than about a 8 nM IC₅₀, and more preferably inhibited by thecompound with less than about a 5 nM IC₅₀.

In some aspects, the methods comprise contacting Ack1 with an effectiveamount of a composition comprising a compound having Formula I, FormulaII, and/or Formula III and a carrier, which may be a pharmaceuticallyacceptable carrier. The composition may comprise any dosage form and/orany excipients, including those described or exemplified herein.Preferably, the biologic activity of Ack1 is inhibited by thecomposition with less than about a 100 nM IC₅₀, more preferablyinhibited by the composition with less than about a 50 nM IC₅₀, morepreferably inhibited by the composition with less than about a 40 nMIC₅₀, more preferably inhibited by the composition with less than abouta 30 nM IC₅₀, more preferably inhibited by the composition with lessthan about a 20 nM IC₅₀, more preferably inhibited by the compositionwith less than about a 10 nM IC₅₀, more preferably inhibited by thecomposition with less than about a 8 nM IC₅₀, and more preferablyinhibited by the composition with less than about a 5 nM IC₅₀.

The invention also features methods for inhibiting the biologic activityof activated cdc42-associated kinase I (Ack1) in a cell or in a tissue.The methods may be carried out in vitro, in vivo, or in situ.

In some aspects, the methods comprise contacting a cell expressing Ack1with an effective amount of a compound having Formula I:

or a pharmaceutically acceptable salt thereof,wherein each of R₁ and R₂ is selected from the group consisting of:R₄—O—, H, and

and

R₃ is

wherein R₄ is a C₁-C₆ alkyl or H;

-   -   R₅ is a C₃-C₈ cycloalkyl such as cyclopropyl, benzyl,

and

-   -   R₆ is F, Cl, or OMe; and    -   R₇ is a C₁-C₃ alkyl.

In some aspects, the methods comprise contacting a cell expressing Ack1with an effective amount of a compound having Formula II:

or a pharmaceutically acceptable salt thereof,wherein R₃ is selected from the group consisting of:

andR₉ is selected from the group consisting of:

wherein R₇ is a C₁-C₃ alkyl; and X is F, Cl, I, Br, or H.

In some aspects, the methods comprise contacting a cell expressing Ack1with an effective amount of a compound having Formula III:

or a pharmaceutically acceptable salt thereof,wherein R₁₀ is selected from the group consisting of:

and R₁₁ is selected from the group consisting of:

benzyl and propyl, wherein R₇ is a C₁-C₃ alkyl; and X is F, Cl, I, Br,or H.

Preferably, the biologic activity of Ack1 is inhibited by the compoundwith an IC₅₀ of about 100 nM or less, more preferably inhibited by thecompound with an IC₅₀ of about 50 nM or less, more preferably inhibitedby the compound an IC₅₀ of about 40 nM or less, more preferablyinhibited by the compound with an IC₅₀ of about 30 nM or less, morepreferably inhibited by the compound with an IC₅₀ of about 20 nM orless, more preferably inhibited by the compound with an IC₅₀ of about 10nM or less, more preferably inhibited by the compound with an IC₅₀ ofabout 8 nM or less, and more preferably inhibited by the compound withan IC₅₀ of about 5 nM or less.

In some aspects, the methods comprise contacting a cell expressing Ack1with an effective amount of a composition comprising a compound havingFormula I, Formula II, and/or Formula III and a carrier, which may be apharmaceutically acceptable carrier. The composition may comprise anydosage form and/or any excipients, including those described orexemplified herein. Preferably, the biologic activity of Ack1 isinhibited by the composition with less than about a 100 nM IC₅₀, morepreferably inhibited by the composition with less than about a 50 nMIC₅₀, more preferably inhibited by the composition with less than abouta 40 nM IC₅₀, more preferably inhibited by the composition with lessthan about a 30 nM IC₅₀, more preferably inhibited by the compositionwith less than about a 20 nM IC₅₀, more preferably inhibited by thecomposition with less than about a 10 nM IC₅₀, more preferably inhibitedby the composition with less than about a 8 nM IC₅₀, and more preferablyinhibited by the composition with less than about a 5 nM IC₅₀.

In any of the methods, the cell may be any cell in which Ack1 isexpressed. The cell may be a cell stably transformed with a nucleic acidencoding Ack1. The cell may be a cell line. The cell may be a cancercell such as a prostate cancer cell, a breast cancer cell, a lung cancercell, a pancreatic cancer cell, an esophageal cancer cell, or a squamouscell carcinoma cancer cell, or may be a neuron. Some examples of cellsinclude LNCaP, LAPC-4, HMEC, 4T1, 293T, and Cos 7 cells.

In some aspects, the invention features compounds. One compound is acompound of Formula I:

or a pharmaceutically acceptable salt thereof,wherein each of R₁ and R₂ is selected from the group consisting of:R₄—O—, H, and

and

R₃ is

wherein R₄ is a C₁-C₆ alkyl or H;

-   -   R₅ is a C₃-C₈ cycloalkyl, benzyl,

and

-   -   R₆ is F, Cl, or OMe; and    -   R₇ is a C₁-C₃ alkyl, provided that when R₁ is H₃CO— and R₂ is        morpholino, R₃ is not cyclopropyl or

where R₇ is methyl, and provided that when R₁ and R₂ are each hydrogen,R₃ is not

where R₅ is benzyl.

In preferred embodiments, R₁ is H₃CO—, R₂ is a morpholinopropoxy group,and R₃ is

where R₅ is a benzyl group or a substituted benzyl group.

One compound is a compound of Formula II:

or a pharmaceutically acceptable salt thereof,wherein R₈ is selected from the group consisting of:

andR₉ is selected from the group consisting of:

wherein R₇ is a C₁-C₃ alkyl; and X is F, Cl, I, Br, or H.

One compound is a compound of Formula III:

or a pharmaceutically acceptable salt thereof,wherein R₁₀ is selected from the group consisting of:

and R₁₁ is selected from the group consisting of:

benzyl and propyl, wherein R₇ is a C₁-C₃ alkyl; and X is F, Cl, I, Br,or H.

Pharmaceutically acceptable salts may be acid or base salts.Non-limiting examples of pharmaceutically acceptable salts includesulfates, methosulfates, methanesulfates, pyrosulfates, bisulfates,sulfites, bisulfites, nitrates, besylates, phosphates,monohydrogenphosphates, dihydrogenphosphates, metaphosphates,pyrophosphates, chlorides, bromides, iodides, acetates, propionates,decanoates, caprylates, acrylates, formates, isobutyrates, caproates,heptanoates, propiolates, oxalates, malonates, succinates, suberates,sebacates, fumarates, maleates, dioates, benzoates, chlorobenzoates,methylbenzoates, dinitromenzoates, hydroxybenzoates, methoxybenzoates,phthalates, sulfonates, toluenesulfonates, xylenesulfonates,pheylacetates, phenylpropionates, phenylbutyrates, citrates, lactates,γ-hydroxybutyrates, glycollates, tartrates, methanesulfonates,propanesulfonates, mandelates, and other salts customarily used orotherwise FDA-approved.

The compounds may be formulated as a composition, for example, with acarrier. Compositions may comprise a compound of Formula I, Formula II,or Formula III, or a pharmaceutically acceptable salt thereof. Thecarrier is preferably a pharmaceutically acceptable carrier.Pharmaceutically acceptable carriers include aqueous vehicles such aswater, alcohol (e.g., ethanol or glycol), saline solutions, dextrosesolutions, and balanced salt solutions, as well as nonaqueous vehiclessuch as alcohols and oils, including plant or vegetable-derived oilssuch as olive oil, cottonseed oil, corn oil, canola oil, sesame oil, andother non-toxic oils. The compositions may comprise one or morepharmaceutically acceptable excipients.

The compositions preferably comprise an effective amount of the compoundsuch as a compound having Formula I, Formula II, Formula III, orpharmaceutically acceptable salt of any of Formula I, II, or III. Thecompositions may be prepared to provide from about 0.05 mg to about 500mg of the compound, or pharmaceutically acceptable salt thereof. Thecompositions may comprise from about 1 mg to about 200 mg of thecompound, may comprise from about 10 mg to about 200 mg of the compound,may comprise from about 10 mg to about 100 mg of the compound, maycomprise from about 50 mg to about 100 mg of the compound, may comprisefrom about 20 mg to about 400 mg of the compound, may comprise fromabout 100 mg to about 300 mg of the compound, and may comprise fromabout 50 mg to about 250 mg of the compound, or pharmaceuticallyacceptable salt thereof.

The compositions may be formulated for administration to a subject inany suitable dosage form. The compositions may be formulated for oral,buccal, nasal, transdermal, parenteral, injectable, intravenous,subcutaneous, intramuscular, rectal, or vaginal administrations. Thecompositions may be formulated in a suitable controlled-release vehicle,with an adjuvant, or as a depot formulation.

Preparations for parenteral administration include sterile solutionsready for injection, sterile dry soluble products ready to be combinedwith a solvent just prior to use, including hypodermic tablets, sterilesuspensions ready for injection, sterile dry insoluble products ready tobe combined with a vehicle just prior to use and sterile emulsions.

Solid dosage forms include tablets, pills, powders, bulk powders,capsules, granules, and combinations thereof. Solid dosage forms may beprepared as compressed, chewable lozenges and tablets which may beenteric-coated, sugar coated or film-coated. Solid dosage forms may behard or encased in soft gelatin, and granules and powders may beprovided in non-effervescent or effervescent form. Solid dosage formsmay be prepared for dissolution or suspension in a liquid or semi-liquidvehicle prior to administration.

Liquid dosage forms include aqueous solutions, emulsions, suspensions,solutions and/or suspensions reconstituted from non-effervescentgranules and effervescent preparations reconstituted from effervescentgranules. Aqueous solutions include, for example, elixirs and syrups.Emulsions may be oil-in water or water-in-oil emulsions.

Pharmaceutically acceptable excipients utilized in solid dosage formsinclude coatings, binders, lubricants, diluents, disintegrating agents,coloring agents, flavoring agents, preservatives, sweeteners, andwetting agents. Enteric-coated tablets, due to their enteric-coating,resist the action of stomach acid and dissolve or disintegrate in theneutral or alkaline intestines. Other examples of coatings include sugarcoatings and polymer coatings. Sweetening agents are especially usefulin the formation of chewable tablets and lozenges. Pharmaceuticallyacceptable excipients used in liquid dosage forms includes solvents,suspending agents, dispersing agents, emulsifying agents, surfactants,emollients, coloring agents, flavoring agents, preservatives, andsweeteners.

Non-limiting examples of binders include glucose solution, acaciamucilage, gelatin solution, sucrose and starch paste. Non-limitingexamples of lubricants include talc, starch, magnesium or calciumstearate, lycopodium and stearic acid. Non-limiting examples of diluentsinclude lactose, sucrose, starch, kaolin, salt, mannitol and dicalciumphosphate. Non-limiting examples of disintegrating agents include cornstarch, potato starch, bentonite, methylcellulose, agar andcarboxymethylcellulose. Non-limiting examples of emulsifying agentsinclude gelatin, acacia, tragacanth, bentonite, and surfactants such aspolyoxyethylene sorbitan monooleate. Non-limiting examples of suspendingagents include sodium carboxymethylcellulose, pectin, tragacanth, veegumand acacia.

Non-limiting examples of coloring agents include any of the approvedcertified water soluble FD and C dyes, mixtures thereof, and waterinsoluble FD and D dyes suspended on alumina hydrate. Non-limitingexamples of sweetening agents include dextrose, sucrose, fructose,lactose, mannitol and artificial sweetening agents such as saccharin,aspartame, sucralose, acelsulfame potassium, and other artificialsweeteners. Non-limiting examples of flavoring agents include syntheticflavors and natural flavors extracted from plants such as fruits andmints, and synthetic blends of compounds which produce a pleasantsensation. Non-limiting examples of wetting agents include propyleneglycol monostearate, sorbitan monooleate, diethylene glycol monolaurateand polyoxyethylene laural ether. Non-limiting examples ofenteric-coatings include fatty acids, fats, waxes, shellac, ammoniatedshellac and cellulose acetate phthalates. Non-limiting examples of filmcoatings include hydroxyethylcellulose, sodium carboxymethylcellulose,polyethylene glycol 4000 and cellulose acetate phthalate. Non-limitingexamples of preservatives include glycerin, methyl and propylparaben,ethylparaben, butylparaben, isobutylparaben, isopropylparaben,benzylparaben, citrate, benzoic acid, sodium benzoate and alcohol.

Elixirs include clear, sweetened, hydroalcoholic preparations.Pharmaceutically acceptable carriers used in elixirs include solvents.Syrups include concentrated aqueous solutions of a sugar, for example,sucrose, and may contain a preservative. An emulsion is a two-phasesystem in which one liquid is dispersed throughout another liquid.Pharmaceutically acceptable carriers used in emulsions may includeemulsifying agents and preservatives. Suspensions may usepharmaceutically acceptable suspending agents and preservatives.Pharmaceutically acceptable substances used in non-effervescentgranules, to be reconstituted into a liquid oral dosage form, includediluents, sweeteners and wetting agents. Pharmaceutically acceptablesubstance used in effervescent granules, to be reconstituted into aliquid oral dosage form, include organic acids and a source of carbondioxide. Sources of carbon dioxide include sodium bicarbonate and sodiumcarbonate. Coloring and flavoring agents may be used in all such dosageforms.

Additional excipients that may be included in any dosage forms include,but are not limited to antimicrobial agents, isotonic agents, buffers,antioxidants, local anesthetic agents, sequestering or chelating agents,analgesic agents, antiemetic agents, and other agents to enhanceselected characteristics of the formulation.

Antimicrobial agents may be cidal or static, and may be antimicrobial,antifungal, antiparasitic, or antiviral. Non-limiting examples ofcommonly used antimicrobial agents include phenols or cresols,mercurials, benzyl alcohol, chlorobutanol, methyl and propylp-hydroxybenzoic acid esters, thimerosal, benzalkonium chloride andbenzethonium chloride. Acidic or basic pH may be used for antimicrobialeffects in some aspects. Non-limiting examples of isotonic agentsinclude sodium chloride and dextrose. Non-limiting examples of buffersinclude phosphate and citrate buffers. A non-limiting example of achelating agent for metal ions is EDTA.

The invention also features methods for treating L-dopa induceddyskinesia in a subject in need thereof. In some aspects, the methodscomprise administering to the subject an effective amount of acomposition comprising a compound having Formula I, Formula H, and/orFormula III and a pharmaceutically acceptable carrier. The compositionmay comprise any dosage form and/or any excipients, including thosedescribed or exemplified herein.

The following examples are provided to describe the invention in greaterdetail. They are intended to illustrate, not to limit, the invention.

Example 1 Experimental Methods

40 ng of recombinant Ack1 was incubated with saturating quantities ofpeptide substrate (Glu-Ala-Ile-Tyr-Ala-Ala-Pro-Phe-Ala-Lys-Lys-Lys) (SEQID NO:1) and in the presence of a compound in Ack1 kinase buffer (20 mMTris pH 7.4, 10 mM MgCl₂, 0.1 mM NaVO₄, 0.5 mM dithiothreitol).[³²P]-γ-ATP was added in a total concentration of 10 μM ATP to initiatethe reaction. Following 30 minute incubation at 30° C., phosphorylatedpeptide substrate was captured by spotting the reaction on P81 cationicfilter paper and unincorporated [³²P]-γ-ATP was removed by washing in asolution of phosphoric acid. Phosphorylated peptide was then quantifiedby autoradiography or by PhosphorImager analysis or by scintillationcounting.

Example 2 Experimental Results

To directly test the kinase selectivity of a large number of establishedkinase inhibitors, high throughput kinase assays were employed against apanel of ^(˜)300 recombinant human protein kinases. An economical methodbased on conventional filter-binding assays was used at a nanoliterscale using radiolabeled ATP that directly measures kinase catalyticactivity toward a polypeptide and/or protein substrate. In contrast toprevious large-scale kinase profiling studies, which typically focusedon clinically relevant kinase inhibitors, compounds that are primarilyused as tools for research were screened. The library of inhibitorscomprises 160 pure compounds known to inhibit kinases representing allmajor protein kinase families, as well as a limited number of compoundsthat were not developed to target protein kinases (FIG. 1A).

The kinase panel tested represents all major human protein kinase. Thedistribution of kinases is shown in FIG. 1B. For simplicity, allcompounds were tested at a concentration of 0.5 μM in the presence of 10μM ATP. The 0.5 μM concentration was chosen despite an average reportedIC₅₀ for these compounds toward their primary targets of 66 nM in orderto capture weaker off-target inhibitory activity.

Each kinase-inhibitor pair was tested in duplicate and results wereexpressed as percent remaining kinase activity compared to solvent(DMSO) control reactions. Disparate replicates were eliminated from theanalysis. FIG. 1C illustrates the reproducibility of the resultingdataset as an x-y plot in which each point represents onekinase-inhibitor pair plotted as the residual kinase activity in onereplicate versus activity in the second replicate, for allkinase-inhibitor pairs in which at least 20% inhibition was observed.

Example 3 New Small Molecule Inhibitors of Ack1

As described above, a large-scale parallel screening of a collection of160 known kinase inhibitors was conducted against a panel of 294-300recombinant human protein kinases. Goals included the identification ofnovel inhibitor scaffolds for new kinase targets and the revelation ofthe target specificities of this panel of research compounds. Eachinhibitor was tested in duplicate at 500 nM against each kinase usingradiolabeled ATP (10 μM total ATP) and a peptide or protein substrate.The resulting dataset comprises almost 100,000 independent functionalassays measuring pairwise inhibition of each kinase by each compound.The data revealed many new targets for these compounds andsimultaneously revealed the selectivity of these compounds among proteinkinases.

The data were analyzed for compounds that inhibit Ack1. Some compounds,like staurosporine, inhibited Ack1 in addition to many other kinases.Therefore, Ack1 inhibitory compounds that showed significant targetselectivity were sought. Inhibitor selectivity was quantitativelyassessed using the Gini coefficient (Piotr P et al. (2007) J. Med.Chem., 50:5773-9; and, Lynette A et al. (2009) J. Chem. Biol. 2:131-51).One compound inhibiting Ack1, compound 1, was unusually selective andshowed significantly greater inhibitory activity against Ack1 thanagainst any other kinase. Interestingly, compound 1 was developed as anATP-competitive inhibitor of the serine/threonine kinase Aurora (Heron NM et al. (2006) Bioorg. Med. Chem. Lett. 16:1320-3) and, indeed, thekinase most potently inhibited after Ack1 (95% inhibition) is Aurora B(75% inhibition). The only other kinase inhibited (by >50%) by 500 nM 1is the non-receptor tyrosine kinase BRK (FIG. 2A).

Compound 1 is an anilinoquinazoline, a highly validated class ofcompounds that target protein kinases. Physicochemical properties ofcompound 1 are consistent with Lipinski rules for drug-likeness(Lipinski C A et al. (2001) Adv. Drug Deliv. Rev. 46:3-26): (<5 hydrogenbond donors (actual value=2), <10 hydrogen bond donors (7), moleculeweight <500 Da (400.43 Da), log P<5 (2.876), and a polar surface area<140 Angstrom2 (85.4 A2). Note that at 400 Da, compound 1 lies at thethreshold of compounds believed capable of passively crossing theblood-brain barrier. Thus, compound 1 is a drug-like molecule thatachieves unusual selectivity for Ack1 over other kinases for anATP-competitive kinase inhibitor.

Example 4

Preliminary Structure-Activity (SAR) Analysis

Ten compounds containing anilinoquinazoline cores or closely relatedstructures were present in the screening library, and many showedsignificant inhibition of Ack1, consistent with a central role of thiscore in contributing to Ack activity. Substitutions of the aniline ringshowed drastic effects on inhibitory activity, suggesting that thestructure of this region is important for activity (data not shown).Dose-response studies of compound 1 revealed an IC₅₀ of 5 nM againstAck1 in vitro (FIG. 2B). By contrast, treatment of HEK293 cells with upto micromolar concentrations of compound 1 did not reduce Ack1autophosphorylation on the activation loop site Tyr284 (data not shown),suggesting poor cell permeability of compound 1. Thus, this Exampleproposed derivatives of compound 1 that inhibit Ack1 in the cellularcontext while retaining its target specificity.

Compound 1 was initially identified as a screening hit for inhibitors ofAurora kinase. It was found that compound 1 showed poor cellularefficacy against Aurora despite good in vitro activity (Heron N M et al.(2006) Bioorg. Med. Chem. Lett. 16:1320-3). Two modifications ofcompound 1 improved cellular activity against Aurora: replacement of thecentral phenyl ring with a pyrimidine and replacement of the methoxygroup at the 7 position in the quinazoline with a3-(1-morpholino)propoxy group (compound 1.1, FIG. 3). To test if thesemodifications would also improve cellular efficacy against Ack1, theywere synthesized (see FIG. 4 for synthetic scheme) in house.

The benzene to pyrimidine substitution reduced Ack1 inhibitoryactivity >100-fold and showed no cell activity (data not shown). Bycontrast, compound 1.1 showed comparable in vitro activity to compound 1(data not shown), but now potently inhibited Ack1 autophosphorylation incells (FIG. 5A). Kinase profiling showed that compound 1.1 inhibitedAck1 more potently than any other kinases and the spectrum of off-targetkinase targets was similar to compound 1, though overall the selectivityof compound 1.1 (by Gini score) was slightly reduced compared tocompound 1 (compare FIGS. 2A and 4B). Thus, substitutions of thequinazoline provide an opportunity to improve cell permeability withoutcompromising inhibitory activity or selectivity.

Compound 1.1 (also known as ZM447439) and a derivative have beenco-crystallized with Aurora kinases, revealing the molecular details ofthe interaction (Heron N M et al. (2006) Bioorg. Med. Chem. Lett.16:1320-3; and, Girdler F et al. (2008) Chem. Biol. 15:552-62). Althoughthe orientation and conformation of the compound in each structure arehighly similar, the compound bound Aurora B as a Type I inhibitor, butAurora A in the Type II mode. In both structures, the orientation of thecompound places the terminal benzamide deep within the ATP-bindingpocket, while the morpholinopropoxy substituent of the quinazoline ringat the other end of the molecule extends out of the pocket, consistentwith the role of this moiety in promoting cell permeability rather thancontributing significantly to kinase binding.

Substitutions of the benzamide have dramatic effects on the activity ofthis scaffold against Aurora (Heron N M et al. (2006) Bioorg. Med. Chem.Lett. 16:1320-3). For example, chloride substitution at C3 of the phenylring enhances potency 30-fold while 4-ethylphenyl reduces activity by^(˜)30 fold. Thus, substituents of the benzamide and quinazoline ringsmodulate Aurora inhibitory activity in vitro and in cells. Because theAck1 construct used in the primary screen comprises only the kinasedomain of Ack1 and a limited additional region C-terminal (amino acids110-476), it is likely that this compound class also targets the Ack1ATP-binding pocket. It is believed that compound 1.1 binds Ack1 in ananalogous manner to its binding to Aurora, and it is believed thatsubstitution of the benzamide and quinazoline rings can be used toenhance relative activity of this compound series against Ack comparedto Aurora and other targets.

The crystal structure of Ack1 has been solved in both an unliganded formand with compounds bound in the ATP-binding pocket. Superposition of thestructure of Ack1 bound to AMPPNP (PDB 1U54) with the structure ofAurora B bound to compound 1.1 (PDB 2VRX) reveals no obvious stericclashes of 1.1 with residues of Ack1 (data not shown). Twenty fiveresidues of Aurora are within six angstroms of 1.1 in the co-structure.Sixty percent of these residues are identical between Aurora and Ack1,consistent with a similar binding mode for the compound to Ack1.Nevertheless, sequence differences between the kinases suggest thepossibility that alterations in the compound could differentially affectkinase binding.

Example 5 Additional Structural Classes of Ack1 Inhibitors

To evaluate other Ack1 inhibitors identified by potential leads, eachcompound was analyzed with regard to its potency in the primary screenand specificity (Gini score). Several of the most potent and specificAck1 inhibitors were anilinoquinazolines related to compound 1. Amongthe remaining promising compounds, compounds 2 and 3 (FIGS. 6 and 7)were selected for further analysis based on their potency against Ack1relative to other kinases and the commercial availability of structuralanalogs.

Compound 2 (also known as PD169316) is a 2,4,5-substituted imidazoledeveloped as a cell-permeable, ATP-competitive inhibitor of p38 MAPkinase. In the screen, compound 2 showed potent inhibition of p38 MAPK(98%), consistent with its reported IC₅₀. In addition, compound 2 showedreasonable activity against Ack1 (54% inhibition) with additionalactivity against a limited number of kinases including casein kinase Iand NLK. The attractive selectivity of this compound, its significantactivity against Ack1, and the availability of structural analogs, leadto focus on this compound as a starting point for the development of asecond Ack1 inhibitor.

Compound 3 (also known as TPCA-1) is a substituted thiophene developedas a cell-permeable, ATP-competitive inhibitor of IκB kinase 2 (IKK-2).Indeed, 91% inhibition of IKK-2 was observed, and importantly, 94%inhibition of Ack1 catalytic activity was observed. Additionaloff-target activities included partial inhibition of the catalyticactivities of FLT3, JAK1/2, RET, and Tyk2.

Example 6 Optimization of Compound 1.1

The goals of the experiments described in this example include thecharacterization of the mechanism of action of compound 1.1, a lead Ack1inhibitor and the optimization of its potency, selectivity, andcell-permeability, for example, a low nanomolar potency in vitro,activity in cells at <200 nM, and >10 fold selectivity over the nextmost potently inhibited kinase in kinase profiling experiments.

A. Mechanism of Action of Compound 1.1.

Rationale and approach. Without intending to be limited to anyparticular theory or mechanism of action, it is hypothesized thatcompound 1.1 binds Ack1 in a similar manner to the manner in which itbinds to Aurora. To test this hypothesis, whether inhibition by 1.1 isATP-competitive will be assessed. In addition, the molecular details ofthe compound interaction will be investigated using collaborativemodeling and crystallographic approaches.

Experimental details. 40 ng of recombinant Ack1 (Carna Biosciences) willbe incubated with saturating quantities of peptide substrate(Glu-Ala-Ile-Tyr-Ala-Ala-Pro-Phe-Ala-Lys-Lys-Lys) (SEQ ID NO:1) and awide range of concentrations of 1.1 in Ack1 kinase buffer (20 mM Tris pH7.4, 10 mM MgCl₂, 0.1 mM NaVO4, 0.5 mM dithiothreitol). Reactions willbe performed and analyzed as done previously (FIG. 2B; (Decon S W et al.(2008) Chem. Biol. 15:322-31)). It is expected that the data willdemonstrate competitive binding with ATP. In parallel, models will begenerated in house to energetically minimize compound 1.1 and analogsdocked into the Ack1 ATP-binding pocket via homology modeling based onthe Aurora B-1.1 co-crystal structure (PDB 2VRX). Crystallography willbe used to determine the precise binding mode of compound 1.1 or analogsto Ack1 and to guide the analysis of the compound analogs discussedbelow.

B. Compound 1.1 SAR: Benzamide Substitutions.

Rationale and approach. The benzamide of compound 1.1 binds deep intothe ATP-binding pocket of Aurora, and substitutions in this ring alterinhibitor potency. It is believed that that substitutions on the phenylring could be used to reduce compound 1.1 activity against Aurora (andother off-target kinases) relative to inhibition of Ack1. To test thishypothesis, an analog library will be synthesized, beginning with asystematic analysis of halides and methoxy groups at para, meta, andortho positions of the phenyl ring. Additional derivatives, describedbelow, will also be purchased and tested in vitro and for their abilityto inhibit Ack1 autophosphorylation in cells. Most potent compounds withcellular activity will be tested for modulation of Erk activation inRas-GRF1-expressing cells to validate this therapeutic approach forL-dopa induced dyskinesia.

Compounds. Compounds 1.2-1.10 (FIG. 3) will be synthesized in house ashas already done for compound 1.1 (FIG. 4), replacing benzoyl chloridein step f with the corresponding substituted benzoyl chloride. Theresults with these compounds (and structural data obtained above) willbe used to guide further SAR exploration. A large variety of substitutedbenzoyl chlorides are available that include additional groups (e.g.,methyl and cyano) added singly and in combination at each ring positionthat will then be tested in an iterative manner (synthesis→biologicalassay and structural modeling→synthesis of new analogs). Compound 1.1analogs replacing the benzamide phenyl ring with cyclopropyl andtrimethylbenzyl groups (compounds 1.11 and 1.12) are also commerciallyavailable and will be obtained and tested.

Biological assays. For dose-response experiments, Ack1 phosphorylationof peptide substrate will be measured in the presence of solvent controlor increasing concentrations of compound as in FIG. 2B. Compounds withIC₅₀<500 nM will be assessed for kinase selectivity by screeningcompounds at 500 nM (10 μM ATP) against their panel of 300 human proteinkinases. Selectivity will be quantified using the Gini score asdescribed above.

Compounds which show more potent inhibition of Ack1 than any otherkinase will be tested for inhibition of Ack1 autophosphorylation incells as in FIG. 5A. Compounds that inhibit Ack1 autophosphorylation incells will next be tested as to whether they inhibit Ras-GRF1 mediatedErk activation in HEK293 cells transiently expressing Ras-GRF1. Cellswill be transfected with Ras-GRF1 and either Ack1 alone, or Ack1 withconstitutively active Cdc42 (Cdc42V12 mutant) to promote Ack1 activity,as previously described (Kiono M J et al. (2000) J. Biol. Chem.275:29788-93). Endogenous Erk activation will be monitored by Westernblotting lysates using phospho-specific antibodies for active Erk. It isbelieved that Ack1 inhibitors will inhibit Erk activation in aRas-GRF1-dependent manner, validating inhibition of LID-relevant pathwayby targeting Ack1.

C. Compound 1.1 SAR: Quinazoline Substitutions.

Rationale and approach. The methoxy and morpholino substituents at thequinazoline C6 and C7 positions of compound 1.1, respectively,critically modulate Aurora inhibitory activity and cell permeability.Without intending to be limited to any particular theory or mechanism ofaction, it is hypothesized that substitutions of the quinazoline maydifferentially affect the activity of this molecule toward Ack1 and itsoff-target kinases to improve selectivity. Compound 1.1 analogs will beassessed using the three assays described above.

Compounds and assays. Compounds 1.13-1.15 will be synthesized in houseby substituting R1 and R2 in the synthesis scheme of FIG. 4. Compound1.16, in which the C6 and C7 quinazoline substituents are hydrogen, iscommercially available. Additional alkyl halides will be explored atthese positions based on the initial studies. Compound activity,selectivity, and cell activity will be tested as above. One goal is toenhance compound selectivity for Ack1 without compromising the importantrole this region plays in cell permeability of the compound.

Alternative approaches and future directions. It is believed that theexperiments to reveal one or more substitutions that increase Ack1inhibitory activity relative to off-target kinases. One goal is >10-foldselectivity. Compounds will be prioritized according to the followingrank order: cellular inhibition of Ack1>in vitro specificity>in vitropotency. This will ensure that resulting compounds remain useful forcellular studies while optimizing specificity and potency. If individualcompound modifications of the benzamide and quinazoline regions improvecompound performance, they will then be combined pairwise through newsyntheses according to the scheme in FIG. 4. If none of the derivativessignificantly improve compound 1.1 selectivity or potency, compoundoptimization will be considered from more one of the more divergentanilionoquinazoline analogs with Ack1 inhibitory activity.

Example 7 Optimization of Compounds 2 and 3

These experiments will test the transition of two additional Ack1compounds into leads with improved potency, specificity, and cellactivity. Commercially available compounds related to two other Ack1inhibitors previously identified will be analyzed to determine ifanalogs can be found with enhanced potency and selectivity toward Ack1and to reveal key features of the SAR.

A. Compound 2 Analogs.

Rationale and approach. The 2,4,5-substituted imidazole, compound 2(FIG. 6), was identified as an inhibitor of Ack1 (54% inhibition) andhas reasonable selectivity for an initial hit. This compound wasdeveloped as a cell-permeable, ATP-competitive inhibitor of p38 MAPK andthe structure of a closely related compound (S8203580) bound to p38 hasbeen reported (Wang Z et al. (1998) Structure 6:1117-28). The pyridinenitrogen makes key contacts with the kinase hinge region and thefluorophenyl group binds within a hydrophobic pocket. The nitrophenylgroup (a 4-(methylsulfinyl)phenyl group in the published structure makescontacts with the phosphate binding region. Because of the nearthree-fold symmetry of this compound family, the orientation of thiscompound in the binding pocket of Ack1 is less clear than forcompound 1. Therefore, SAR studies of this compound family will exploitdiverse commercially available derivatives of 2 in in vitro andcell-based assays.

Experimental details. Whether inhibition of Ack1 by 2 is competitivewith ATP will be tested as described in example 6 above. Next, whetherthe pyridine nitrogen plays an important role in kinase binding as itdoes for p38 MAPK, will be assessed by testing a compound in which thisring is replaced by phenyl (not shown, commercially available). It isbelieved that this will significantly reduce activity but, if not, alarge number of commercially available compounds with phenyl at thisposition will be screened to validate this scaffold as a better lead.

If the pyridine is important, compounds in which the substituted phenylsat position 2 and 5 of the imidazole are replaced will be tested.Compounds 2.1-2.3, and 2.7 will test a variety of substituted phenylgroups at imidazole ring carbon 2 in the context of the 4-fluorophenylsubstituent at carbon 5. Compounds 2.4-2.6 and 2.8 will elucidate theactivity of compounds in which the 4-fluorophenyl group is replaced withother substituted phenyls. All compounds will be tested initially at 500nM against Ack1 catalytic activity in vitro as above. Most potentderivatives will be subjected to complete dose-response analysis, kinaseprofiling, and cell-based assays as above.

B. Compound 3 Analogs

Rationale and approach. The substituted thiophene, compound 3, showed94% inhibition of Ack1 and reasonable selectivity. It was developed as apotent (18 nM), ATP-competitive, and cell-permeable inhibitor of IKK-2.No structural data concerning the binding mode of this compound to IKK-2has been reported. Five commercially available 3-thiophenecarboxamidesrelated to compound 3 have been identified (FIG. 7), and the importanceof the substitutents at the 2 and 5 positions of the thiophene will besystematically tested.

Compounds and assays. Compound 3.1 differs from compound 3 in the lossof the fluorine substituent on R2, compound 3.2 replaces the urea withan acetamide, and compounds 3.3-3.5 extend the urea with either para,meta, or ortho fluorphenyl. Compounds will be assessed and prioritizedas above.

Alternative approaches. It is believed that the SAR studies will revealmore potent and/or more selective inhibitors of Ack1 than the initialhit molecules. Nevertheless, if this is not the case, kinase profilingof these diverse compounds (against 300 protein kinases) as proposed mayreveal novel inhibitors for kinases other than Ack1 as a byproduct.Unusually potent or specific compounds, or compounds targeting kinasesfor which no small-molecule inhibitors currently exist, will be offeredto experts in their particular targets for further characterization.

The invention is not limited to the embodiments described andexemplified above, but is capable of variation and modification withinthe scope of the appended claims.

-   N-[4-[[6-Methoxy-7-[3-(4-morpholinyl)propoxy]-4-quinazolinyl]amino]phenyl]benzamide

-   4-[5-(4-fluorophenyl)-2-(4-nitrophenyl)-1H-imidazol-4-yl]-pyridine

-   2-[(Aminocarbonyl)amino]-5-(4-fluorophenyl)-3-thiophenecarboxamide

We claim:
 1. A compound of Formula I:

or a pharmaceutically acceptable salt thereof, wherein each of R₁ and R₂is selected from the group consisting of: R₄—O—, H,

and R₃ is

wherein R₄ is a C₁-C₆ alkyl or H; R₅ is a C₃-C₈ cycloalkyl, benzyl,

and R₆ is F, Cl, or OMe; and R₇ is a C₁-C₃ alkyl, provided that when R₁is H₃CO— and R₂ is morpholino, R₅ is not cyclopropyl or

where R₇ is methyl, and provided that when R₁ and R₂ are each hydrogen,R₃ is not

where R₅ is benzyl.
 2. The compound of claim 1, wherein R₁ is H₃CO—; R₂is

and R₃ is


3. The compound of claim 1, wherein R₁ is H₃CO—; R₂ is

and R₃ is


4. The compound of claim 1, wherein R₁ is H₃CO—; R₂ is

and R₃ is


5. The compound of claim 1, wherein R₁ is

R₂ is H₃CO—; and R₃ is


6. The compound of claim 1, wherein R₁ is H; R₂ is H; and R₃ is


7. The compound of claim 1, wherein R₁ is H₃CO—; R₂ is

and R₃ is


8. A composition, comprising the compound of claim 1 and a carrier. 9.The composition of claim 8, wherein the carrier is a pharmaceuticallyacceptable carrier.
 10. A composition, comprising the compound of claim2 and a carrier.
 11. The composition of claim 10, wherein the carrier isa pharmaceutically acceptable carrier.
 12. A composition, comprising thecompound of claim 3 and a carrier.
 13. The composition of claim 12,wherein the carrier is a pharmaceutically acceptable carrier.
 14. Acomposition, comprising the compound of claim 4 and a carrier.
 15. Thecomposition of claim 14, wherein the carrier is a pharmaceuticallyacceptable carrier.
 16. A composition, comprising the compound of claim6 and a carrier.
 17. The composition of claim 16, wherein the carrier isa pharmaceutically acceptable carrier.
 18. A composition, comprising thecompound of claim 7 and a carrier.
 19. The composition of claim 18,wherein the carrier is a pharmaceutically acceptable carrier.